A Middle Pleistocene wolf from central Italy provides insights on the first occurrence of Canis lupus in Europe

Here, we describe a partial cranium of a large canid dated at 406.5 ± 2.4 ka from the Middle Pleistocene of Ponte Galeria (Rome, Italy). The sample represents one of the few Middle Pleistocene remains of a wolf-like canid falling within the timeframe when the Canis mosbachensis–Canis lupus transition occurred, a key moment to understand the spread of the extant wolf (Canis lupus) in Europe. CT-based methods allow studying the outer and inner cranial anatomy (brain and frontal sinuses) of a selected sample of fossil and extant canids. Morphological and biometric results allowed to: (I) ascribe the cranium from Ponte Galeria to an adult Canis lupus, representing the first reliable occurrence of this taxon in Europe; (II) provide the content for a biochronological revision of the Middle Pleistocene record of European wolves.

www.nature.com/scientificreports/ Outer cranial anatomy. The specimen PF-PG1 (Figs. 3,4) consists of a partial braincase divided into four fragments of different size. The surface of the fragments is covered by a thin and irregular encrusting patina of volcanic ash, light grey in color. The coarse volcanic sediment is mostly located within the tympanic bullae, while it is less abundant inside the frontal bone. The specimen has completely fused cranial sutures, which is compatible with the adult condition, and it shows no signs of bone alterations or transport (weathering or abrasion). The postorbital constriction is wide (even if preserved only on the left side), the slightly marked left temporal line ending posteriorly to a long and well-developed sagittal crest. The neurocranium is fairly inflated in dorsal view. The frontals are convex and elevated, whereas the sagittal crest is quite robust and the projection posterior end exceeds the occipital condyles in lateral view. The retroarticular process is robust as well the mastoid one, whereas the opening of the acoustic meatus is oval with antero-ventral to postero-dorsal major axis. Posteriorly, the braincase is rounded and has a rough surface, with a marked nuchal crest. The foramen magnum is oval and elongated laterally, the occipital condyles are laterally and dorsally projected and the nuchal tubercle is weak. The tympanic bullae are partially broken. They are antero-medially to postero-laterally elongated, their anterior borders are aligned with the retroarticular processes, and their medial walls are parallel in ventral view. PF-PG1 shows frontal bones slightly convex in lateral view, similarly to those of other fossil and extant specimens of C. lupus, but differing from the condition of C. mosbachensis, where the frontals are less prominent 10,13 (Supplementary Fig. S3). The medial wall of the tympanic bullae is divergent in the Ponte Galeria specimen, similarly to those of C. lupus and C. mosbachensis 13 . Biometric comparison. Cranial measurements of PF-PG1 (Fig. 5, Supplementary Table S3) falls within the range of extant C. lupus italicus for all the considered variables. In the GMB-HOT plot, PF-PG1 is larger than specimens from Lunel-Viel and Grotta Romanelli, ranking close to those of Covoli di Velo, Grotta Ladrenizza and extant C. lupus italicus (Fig. 5). A similar arrangement is observable in the GMB-GBOC plot, except for a single specimen from Lunel-Viel which is nearly as large as those from Covoli di Velo, Grotta Ladrenizza and Ponte Galeria. In GBOC-HOT, GBOC-GBFM and HFM-GBFM plots, C. lupus from Grotta Romanelli and C. mosbachensis from Cerè fall outside or within the lower portion of the range of extant C. lupus italicus (Fig. 5).
The specimen of C. lupus maximus from Grotte de Jaurens is the largest among the analyzed sample and falls outside the range of the extant Italian wolf. In GDAB-GBFM and GDAB-GBOC plots (Fig. 5) 6) extending from the antorbital constriction up to the fronto-parietal suture. In dorsal view, its anterior portion-between the preorbital constriction and the zygomatic process of the frontal-is laterally expanded, lobed and crossed by a deep transverse groove, while the caudal one-between the zygomatic process of the frontal and the frontoparietal suture-is narrower, less lobed, with a compact and smooth surface (Fig. 6). In lateral view, the sinus is triangularly shaped and quite domed. The frontal sinus as it appears in Fig. 6, is 43.7 mm long and 19.1 mm wide (the comparative list of measurements is reported in Supplementary Table S4), reaching almost half the length of the brain endocast. In dorsal view, it covers the whole anterior portion of the brain extending to the cruciate sulcus, while laterally it completely covers the orbital gyrus. The frontal bone of the Ponte Galeria specimen contains a large frontal sinus, morphologically similar to those of fossil (Grotta Romanelli) and extant C. lupus (e.g., C. lupus italicus, C. lupus lycaon, C. lupus baileyi) (Fig. 6). According to Curtis and Van Valkenburgh 37 , the frontal sinuses of C. lupus are the largest among all living canids, representing a peculiarity of the species. Frontal sinuses of L. pictus are strongly lobed, reduced in size and placed between the preorbital and postorbital constriction. In dorsal view they do not reach the fronto-parietal suture nor the cruciate sulcus of the brain, while in lateral view they do not fully cover the orbital gyrus (Fig. 6). Considering canids of smaller sizes, such as C. aureus, C. latrans and Lupulella mesomelas, their frontal sinuses are strongly reduced with a simplified morphology. They are dorsally flat with a smooth surface, and they never reach the fronto-parietal suture nor the cruciate sulcus of the brain (Fig. 6).
Brain. Processing CT-images, we obtained a partial brain endocast (Fig. 7) consisting of an upper left hemisphere with clearly visible convolutions, and a portion of the right one, which shows a smoother surface and less marked sulci. The olfactory bulbs, most of the anterior left hemisphere, the entire basal portion of the cerebrum and almost all the cerebellum are missing. The brain endocast, as it appears in Fig. 7, is 74.1 mm long and  Table S5). In dorsal view, a poorly marked longitudinal fissure divides the telencephalon into two almost symmetrical hemispheres with convolutions represented-from front to back-by the orbital, prorean, lateral, sigmoid, coronal, endolateral, ectolateral, suprasylvian and ectosylvian gyri, including the respective sulci (for the complete list of the brain www.nature.com/scientificreports/ convolutions see Fig. 7). In dorsal and lateral views, the brain is morphologically similar to the sample from Grotta Romanelli and to those of the extant C. lupus italicus, C. lupus lycaon, C. lupus baileyi and C. simensis. In  www.nature.com/scientificreports/ these specimens, the frontal pole cortex is antero-posteriorly elongated, the orbital gyrus is laterally expanded forming a quite developed "bump" bounded by the intraorbital sulcus, the prorean gyrus is long and bilaterally constricted. Moreover, the orbital region is characterized by the presence of three main sulci: the prorean sulcus, the intraorbital sulcus and a third sulcus. According to Lyras and Van der Geer 38 the third sulcus is considered typical of C. lupus, C. simensis and C. rufus. In the remaining species of our comparative sample the frontal pole is less elongated, and the orbital gyri are delimited in C. latrans and L. pictus by the prorean and intraorbital sulcui, while in C. aureus and Lupulella mesomelas only by the prorean sulcus. On the left hemisphere of extant C. lupus italicus, C. lupus lycaon and L. pictus a small dimple is detectable on the anterior portion of the coronal gyrus which is missing in the rest of the sample, including the specimen from Ponte Galeria and the fossil wolf from Grotta Romanelli and C. lupus baileyi. This feature was reported by Radinsky 39 and Lyras 40 only in C. lupus, Cuon alpinus and L. pictus and according to the authors it seems to be missing in all other canid species.
In Grotta Romanelli and Ponte Galeria specimens, the surface irregularities of the brain endocasts do not allow the identification of this character, which however is not recognizable in C. lupus baileyi despite the good quality of the brain model. The brain volume of the specimen from Ponte Galeria has not been measured due to the incompleteness of the endocast.

Discussion
The wolf from Ponte Galeria dated at ca. 407 ka represents the largest cranial remain of a Middle Pleistocene canid known to date in Europe. Due to the fragmentary nature of the specimen, CT-based methods have been used to acquire a wider set of morphological data, including those offered from the brain and frontal sinuses. The sample from Ponte Galeria shows the following wolf-like features: (I) elevated frontal bones, (II) well-developed sagittal and occipital crests, (III) large and domed frontal sinus which extends to the fronto-parietal suture reaching the cruciate sulcus of the brain, (IV) rostral pole of the brain antero-posteriorly elongated with the orbital gyrus laterally expanded. In size, the specimen PF-PG1 is larger than C. mosbachensis, matching the values of moderately large individuals of C. lupus (Fig. 5), while morphologically it is almost indistinguishable from the extant wolf and it appears more robust compared to C. mosbachensis (Supplementary Fig. S3). In the latter species, the frontals are less swollen than in Canis lupus, suggesting a lower extension of the frontal sinuses. Strongly pneumatized and dorsoventrally expanded frontals are considered a distinctive trait of the wolf, since this species possesses the largest frontal sinuses among all extant canids 37 . Although frontal sinuses of C. mosbachensis have never been formally described, Tedford et al. 12 indicates that in this species the frontal sinuses are large, but they never reach the fronto-parietal suture-character 34(2) of their phylogenetic analysis-as instead occurs in the wolf-character 34 (3). Large sinuses have been found throughout our sample of Late Pleistocene and extant C. lupus (Fig. 6), thus confirming the consistency of the character 34(3) scored by Tedford et al. 12 for C. lupus. In the sample of Ponte Galeria, the left frontal sinus is large, domed and extends up to the fronto-parietal suture, perfectly corresponding in size and shape to those of the wolf (Fig. 6). The neuroanatomy of fossil canids was explored by few authors 4,38-41 and studies on extensive fossil specimens have never been performed. Additionally, no brain endocasts of Middle Pleistocene wolf-like canids have  www.nature.com/scientificreports/ been documented in Europe. As a result, our knowledge of the brain anatomy of C. mosbachensis and early C. lupus is limited. The partial brain endocast of PF-PG1, with an age of ca. 407 ka, fills this gap and appears as a brain of an extant wolf. The overall size, the antero-posteriorly elongated frontal pole cortex, the convolution pattern and the presence of a marked orbital gyrus bounded by the three sulci, one of which-the third sulcus, is considered typical of C. lupus, C. simensis and C. rufus 38 , point out the morphological affinities with the wolf (Fig. 7). Studies on the brain anatomy of social carnivorans evidenced a possible relation between the development of the frontal pole cortex and sociality. For instance, in domestic dogs the precruciate and prorean gyri are involved in social action and interaction 42 , the prorean gyrus has been also linked to the emergence of pack structure in canid evolution 43 , and in extant Crocuta crocuta the expansion of the frontal cortex has been linked to increased sociality 44 . In PF-PG1 the overall development of the rostral portion of the brain, including the orbital, precruciate and prorean gyri, matches the proportions of C. lupus and C. simensis, which are both able to form packs spanning from 3 to more than 13 individuals, depending on food availability 45 . Hence the morphological resemblance with PF-PG1 allows to assume that such pack structures were already present in Middle Pleistocene wolves of Europe, although additional paleoneurological studies on the frontal cortex of fossil canids are required to consolidate this hypothesis.
Considering the above, we ascribe the Ponte Galeria specimen to an adult Canis lupus. We excluded the attribution to Canis mosbachensis because in this hypothesis the specimen from Ponte Galeria would represent the largest and most robust cranial remain of C. mosbachensis ever recorded, contradicting what is generally claimed for this species, namely that it is slender and of a size comparable to that of the extant subspecies C. lupus pallipes 11 . It could be argued that size alone should not be taken as a valid criterion for specific distinction, especially considering the huge biometric and morphological variability observed between different populations of extant C. lupus. The Mosbach wolf has often been considered a chronosubspecies of the wolf, C. lupus mosbachensis 23,46 , a view that only after the description of the large sample from the Early Pleistocene of Untermassefeld became less widespread 11,12 (Supplementary Note 1). Emerging data on dental remains of Middle Pleistocene canids from Mediterranean Europe, further support a close ancestor-descendent relation with C. lupus and confirm the difficulties in identifying valuable diagnostic characters for C. mosbachensis 4,10,47 . In wide-ranging mammals like C. lupus the body size is known to vary according to several factors, for instance reflecting ecomorphological adaptations to different environments or climatic conditions, the availability of trophic resources, and competition with sympatric species. Investigating size fluctuations through time adds a dimension of complexity while not excluding factors that operate across other scales. Increasingly, many studies recognized differences between trends advocated for explaining the general evolution of large mammal species and fluctuations that become evident at a closer geographical scale, as for instance documented in Europe for the spotted hyena 48 , the wild boar 49 and the horse 50 . As for C. lupus, differences in size fluctuations through time were noted even comparing the geographically close fossil records of France and Italy 51 . However, recognizing the complex interplay of these factors in explaining the observed variation in C. lupus underlines a striking contrast with the lower dimensional variability of C. mosbachensis. Samples assigned to the Mosbach wolf span in chronology about twice the time from the FO of C. lupus to the present day, and yet none of them includes outliers of a large size comparable to that of subsequent populations of C. lupus 11,19,52 . The problem arises when approaching the "transitional" period of the late Middle Pleistocene, with forms of intermediate size such as C. lupus lunellensis that can be hardly significantly separated on a biometrical ground 10,19,22,23 . Although it cannot be completely ruled out that some Middle Pleistocene forms of C. mosbachensis may have reached a large body size, a significant preservation bias is unlikely considering the abundance of dental remains in the early Middle Pleistocene 10,19,53 . A potential explanation may rest on the faunal turnover occurred at the time of the FO of C. lupus, the Galerian-Aurelian transition, which saw the extinction of several large-sized carnivorans 14 . This renewal may have open new niches for an opportunistic predator to be exploited, hence eventually promoting local adaptations in size in different ecological scenarios.
The Ponte Galeria wolf is dated close to the Mid-Brunhes Event (ca. 424 ka; MIS 12-11 transition), which marks the end of the Early-Middle Pleistocene Transition and the consolidation of the glacial cycles ruled by a 100 kyr periodicity 54 . After this transition an increase in the amplitude of interglacials is recorded with one of the longest and warmest interglacial occurring right after the extreme MIS 12 glacial, during MIS 11 55 . In central Italian Peninsula, MIS 11 seems to have been characterized by a marked seasonality, as suggested by the analysis of the diets of fossil herbivorous ungulates from Fontana Ranuccio (408 ± 10 ka; Anagni, Central Italy) 56 , and by pollen-based climate reconstructions of Combourieu-Nebout et al. 57 , in which the lowest winter temperatures of all the examined Middle Pleistocene interglacials are inferred during MIS 11 (in the Boiano locality; Campobasso, Central Italy). The earliest dispersal of C. lupus is one of the bioevents selected to define the beginning of the Aurelian Mammal Age of the Italian large mammal biochronological scale 14 . Two additional taxa appeared during the Aurelian, Megaloceros giganteus and Ursus spelaeus, but studies of the last decades suggest an older diffusion, occurred during MIS 11 and MIS 13, respectively 29,58 . After Gliozzi et al. 14 , many studies added important information on the timing of dispersals or extinction of several large mammals 59,60 . During MIS 13-10, multiple key bioevents occurred in Europe favoring the spread of Bos primigenius 32 , Equus hydruntinus 61 and Dama clactoniana 62,63 , evidencing how terrestrial ecosystems were strongly affected by the Mid-Brunhes Event. Considering the large turnover recorded during the Mid-Brunhes Event, it is thus possible that the severe glacial conditions of MIS 12 may have played a role in triggering the spread of large-sized wolves, and/or that the warmer and seasonal conditions started after MIS 12 favored the establishment of viable populations. The presence of morphologically modern wolves in North America in the late Early Pleistocene 12 and the abundance of C. mosbachensis in East Asia during the early Middle Pleistocene 64 suggest a dispersion of C. lupus into Europe, rather than a local origin of a modern phenotype. In this scenario large-sized wolves may have dispersed into Europe encountering or even interbreeding with populations of C. mosbachensis previously inhabiting the region. The impoverishment in the carnivoran guild may have favored the spread of these newcomers 14  www.nature.com/scientificreports/ them to occupy a wider range of niches compared to C. mosbachensis. Considering dental measurements, largesized wolves are documented since MIS 8-7 in Europe 52,53 , but the Ponte Galeria cranium supports an earlier arrival of C. lupus during MIS 11, ca. 400 ka. To better understanding the tempo and mode of the acquirement of the modern phenotype along and before the C. mosbachensis-C. lupus transition, and possibly of the origin of the wolf, CT-based studies of Early to Middle Pleistocene wolf-like canids are needed. Concluding, PF-PG1 attests the presence of modern wolf-like morphologies in Europe at 406.5 ± 2.4 ka, suggesting a climate-induced turnover between the latest Early to early Middle Pleistocene forms of C. mosbachensis and those succeeding referable to C. lupus. It remains to be clarified whether C. mosbachensis represents a reliable taxonomic entity or it should rather be considered an early morphotype of C. lupus.

CT-scanning and digital restoration. Tomographic images of Canis lupus lycaon, Canis lupus baileyi and
Canis simensis were downloaded from MorphoSource.org (ark:/87602/m4/M113796, ark:/87602/m4/M114099, ark:/87602/m4/M113802). Tomographic images of the rest of the sample were taken using a Philips Brilliance CT 64-channel scanner at M.G. Vannini Hospital (Rome). The cranial fragments were scanned together in the coronal slice plane from front to back. The slice thickness is 0.67 mm with an interslice space of 0.33 mm. No skulls of Canis mosbachensis were available for the examination of the frontal sinuses and the brain. CT image processing and measurements of the frontal sinuses (Supplementary Table S4) and brains (Supplementary Table S5) have been acquired using Mimics 21.0. Each fragment of the studied braincase has been digitally reconnected matching the complementary fragments. The high correspondence of the bone margins and the use of 3D reference skulls of both extant and fossil wolves, allowed to obtain a restored 3D model of the Ponte Galeria specimen. The process of fragment alignment was carried out with ZBrush 4R6. The restored model generated during the current study is available in the MorphoSource repository (ark:/87602/m4/408336).
Analytical methods. Before CT scan the specimen has been sampled. The volcanoclastic matrix occurring in the internal and external parts of the skull was gently removed from the bone using a thin blade. The sediment was split at the binocular, and volcanic components were picked and mounted as polished and carbon-coated thin section. Texture was observed at the Department of Earth Sciences, Sapienza University of Rome, using the polarized optical microscope and the FEI Quanta 400 electron microscope. EMP analyses of glasses were carried out at the CNR-Istituto di Geologia Ambientale e Geoingegneria di Roma, with a Cameca SX50 electron microprobe equipped with five wavelength dispersive spectrometers (WDS). Quantitative analyses were performed using 15 kV accelerating voltage and 15 nA beam current. As standards we employed metals for Mn and Cr, Jadeite for Na, Wollastonite for Si and Ca, Orthoclase for K, Corundum for Al, Magnetite for Fe, Rutile for Ti, Periclase for Mg, F-apatite for P, phlogopite for F, potassium chloride for Cl, barite for S. Counting times for all elements were 20 s on peak and half time on both backgrounds. Light elements (Na, K) were counted first to prevent loss by volatilization. The PAP correction method was used. Glasses were analyzed using a beam diameter of 15 µm to minimize alkali loss. In order to evaluate the accuracy of the analyses, repeated analyses of three international secondary standards (Kakanui augite, Iceladic Bir-1 and rhyolite RLS glasses from USGS) were made prior to any series of measurements. The mean precision from the standard value was about 1% for SiO2, 2% for Al2O3, 5% for K2O, CaO and FeO, and 8-10% for other elements. Moreover, the analytical precision (2 sigma error) is ≤ 1% for elements in the concentration range > 10 wt.% oxide, 5% for elements in the range 2-10 wt.% oxide and better than 10% for elements in the range 0.5-2 wt.% oxide. www.nature.com/scientificreports/